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In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source.
Journal article

In Meso In Situ Serial X-Ray Crystallography (IMISX): A Protocol for Membrane Protein Structure Determination at the Swiss Light Source.

  • Huang CY Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, Villigen-PSI, 5232, Switzerland. Chia-Ying.Huang@psi.ch.
  • Olieric V Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, Villigen-PSI, 5232, Switzerland.
  • Caffrey M Membrane Structural and Functional Biology (MS&FB) Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
  • Wang M Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, Villigen-PSI, 5232, Switzerland.
  • 2020-03-01
Published in:
  • Methods in molecular biology (Clifton, N.J.). - 2020
IMISX
In situ
Lipid cubic phase
Membrane proteins
Mesophase
Microcrystals
Serial crystallography
Synchrotron
X-ray
X-ray free-electron lasers
English The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation. The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe an IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection, and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets-the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G-protein-coupled receptor CCR2A.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://sonar.ch/global/documents/1024
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